The Mechanism Of Hypoxia Regulating C18-4 Self-renewal Of Spermatogonia Stem Cells Through Iron Metabolism-related Proteins

Objective:to study the changes of self-renewal ability and iron metabolism related protein expression of mouse spermatogonia stem cells C18-4 cultured in vitro in simulated hypoxic environment.Methods:firstly,the temporal and spatial expression profiles of HIF-α and iron metabolism proteins in human and mouse testes were drawn by single cell sequencing.Then the model of mouse spermatogonia stem cells(C18-4 cell line)cultured in vitro was established to simulate the hypoxia environment.The effect of hypoxia on the proliferation of mouse spermatogonia stem cells was detected by CCK-8 method and EdU method,and the expression of HIF-1 a protein and iron metabolism related protein in the cells was detected by Western blotting.At the same time,the mRNA expression of iron death-related proteins was detected by qPCR method.The purpose of this study was to clarify the effects of hypoxia on the self-renewal of C18-4 and the expression of iron metabolic proteins in spermatogonia stem cells,and to explore its possible mechanism.Results:1.Under normoxic conditions,C18-4 was round or oval under microscope,attached to petri dishes and proliferated to form colonies.In addition,with the increase of culture time,the number of SSCs colonies and the number of SSCs colonies also increased.2.Under the condition of simulated hypoxia,the number of C18-4 cell colonies and the number of cells in each colony decreased,and with the increase of hypoxia culture time in vitro,the number of cell colonies decreased significantly compared with the control group,and the density gradually decreased,and the morphology gradually became irregular with the increase of the concentration of cobalt chloride(CoCl2).The proliferation energy of C18-4 cells cultured under 3.CoCl2 simulated hypoxia was detected by CCK-8 method at 0h,24h,48h and 72h.With the increase of time,the ability of cell proliferation decreased gradually.4.C18-4 cells were treated with CoCl2 at concentrations of 50.0,100.0,150.0 and 200.0 μ mol/L CoCl2 for 24 h,respectively.The proliferation ability of C18-4 cells decreased gradually with the increase of CoCl2 concentration(P<0.001).After C18-4 cells were treated with 5.CoCl2 for 48 hours,EdU results showed that the proliferation ability of C18-4 cells in hypoxia group was significantly lower than that in control group.6.C184 cells were treated with 200 μmol/L CoCl2 for 24 h,48 h and 72 h,respectively.The expression of HIF-1 a protein was observed by Western blotting.Compared with the control group,the expression of HIF-1 a protein in hypoxia group increased at 24 h and 72 h,and the expression of HIF-1 a protein in C18-4 cells treated with 200 μ mol/L CoCl2 for 72 h was significantly higher than that in control group(P<0.0001).7.C18-4 cells were treated with 50,100,150,200 μ mol/L CoCl2 for 24 hours,and the expression of HIF-1 a protein was observed by Western blotting.Compared with the control group,the expression of HIF1 α protein in the hypoxia group(100,150 μ mol/L CoCl2 group)was significantly higher than that in the control group(P<0.001),and the expression level of HIF-1 a protein in the hypoxia group was significantly higher than that in the control group(P<0.001).8.When C18-4 cells were treated with 200 μ mol/L CoCl2 for 24 hours,the expression level of FPN protein in hypoxia group was significantly lower than that in control group,but the expression level of FTH and FTL protein in hypoxia group was significantly higher than that in control group.9.The levels of GPX4,Acsl4,Sat-1 and DJ-1 in the cells of the two groups were detected by fluorescence quantitative PCR and their relative expressions were calculated.Compared with the control group,the levels of GPX4 and Acsl4mRNA did not change significantly,but the level of Sat-1mRNA increased and the level of DJ-1mRNA decreased significantly(P<0.001).10.The content of MDA in C18-4 cells can reflect the level of lipid peroxidation,compared with the control group,the content of MDA in hypoxia group increased significantly,and the difference was statistically significant.11.C18-4 cells cultured under simulated hypoxia were treated with antioxidants and iron death inhibitors.After 24 hours culture,the number and density of C18-4 cells in NAC experimental group were significantly higher than those in hypoxia control group(200 μmol cobalt chloride treatment for 24 hours).However,there was no significant change in the number and density of cells in Uridine experimental group and Fer-1 experimental group.Conclusion:the self-renewal ability of C18-4 cells decreased and the protein expression of hypoxia-inducible factor-1 α increased in C18-4 cells under hypoxia.The changes of iron metabolism related proteins,which are the downstream regulatory molecules of hypoxia inducible factor in C18-4 cells cultured in vitro under hypoxia condition,suggest that the imbalance of iron homeostasis in spermatogonia stem cells leads to the accumulation of lipid peroxides,which may be a molecular mechanism of hypoxia inducible factor regulating the self-renewal ability of spermatogonia stem cells.This study provides a new theoretical basis for further study of the etiology of male infertility and a new experimental basis for further exploring the molecular mechanism of self-renewal of spermatogonia stem cells.